Antibody 4G8B4B12

ABSTRACT

The invention relates to a monoclonal antibody specifically binding to the human FLT3/FLK2 receptor protein. The invention further relates to hybridoma cells producing such an antibody, as well as to a method for generation of such hybridoma cells. The monoclonal antibody is the antibody produced and released by hybridoma cells deposited under No. DSM 2249 at the German Collection of Microorganisms and Cell Cultures (DSMZ) and designated as 4G8B4B12.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a monoclonal antibody directed against thehuman tyrosine kinase receptor FLT3/FLK2.

The tyrosine kinase receptor FLT3/FLK2 plays an important role in earlyhematopoiesis. It naturally occurs in blood stem cells and earlylymphoid and myeloid blood progenitor cells and interacts with a growthfactor FLT3-ligand (FL), that particularly stimulates the recruitmentand proliferation of these cells (Matthews et al., Cell 65:1143, 1991;Small et al., Proc. Natl. Acad. Sci. USA 91:459, 1994; Lyman et al.,Blood 83:2795, 1994; Muench et al., Blood 85:963, 1995; Hannum et al.,Nature 368:643, 1994; Hirayama et al., Blood 85:1762, 1995).

2. Description of the Related Art

The indirect detection mediated by an antibody specifically binding tothe receptor represents a safe and quick method to qualitatively andquantitatively detect membrane bound receptors. The specific antibodycan be labeled either directly or indirectly, using this label foridentification and quantitative determination of the antibody or thebound receptor, respectively. Particularly, fluorescent dyes orradioactive agents can be used as labels.

Such an antibody can also be coupled to special therapeutically activeagents and therefore renders possible a targeted cellular treatment,which is particularly required for the treatment of tumorous diseases.

Antibodies directed against FLT3/FLK2 receptor protein have already beendescribed, however, these antibodies are directed against the murineFLT3/FLK2 receptor protein. Therefore, lacking the required specificity,these antibodies are not suitable for the treatment of human cells.

A monoclonal antibody binding specifically to the native unmodifiedhuman FLT3/FLK2 receptor protein, is produced and released by hybridomacells that were deposited on Dec. 6, 1997 at the InternationalDepository Authority DSMZ-Deutsche Sammlung Von Mikroorganismen UndZellkulturen Gmbh (German Collection of Microorganisms and CellCultures, Ltd. (DSMZ)), Mascheroder Weg 1b, D-38124 Braunschweig Germanyunder No. DSM ACC2248 at the German Collection of Microorganisms andCell Cultures Ltd. (DSMZ) in accordance with the provision of theBudapest Treaty. It has been given the designation BV10A4H2. Thisantibody is subject matter of German Patent DE 196 08 769 titled"antibody BV10A4H2".

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a furtherantibody specifically binding to the native unmodified human FLT3/FLK2receptor protein and which is available in practically unlimitedamounts.

This object is achieved by providing a monoclonal antibody bindingspecifically to the native unmodified human FLT3/FLK2 receptor proteinand being produced and released by hybridoma cells that were depositedunder No. DSM ACC2249 at the German Collection of Microorganisms andCell Cultures Ltd. (DSMZ) in accordance with the provisions of theBudapest Treaty. The antibody has been given the designation 4G8B4B12.

With the antibody according to the invention, a monoclonal antibody hasbeen provided for the first time that is reproduceable in a standardizedmanner and can thus potentially be produced in unlimited amounts andthat binds specifically to a respective special extracellular epitope ofthe human receptor protein FLT3/FLK2.

The antibody according to the invention permits a targeted detection andmodulation of cells comprising an extracellular domain of the humanFLT3/FLK2 receptor protein. It therefore provides to physicians andresearch personnel a sofar unique and variably applicable means for onthe one hand detecting such cells, both in cell culture and in thepatient's organism, and on the other hand manipulating such cells, ifdesired, either by means of the antibody as such, or by specificreagents coupled to it.

The invention further relates to hybridoma cells producing a monoclonalantibody directed against the FLT3/FLK2 receptor protein and beingdeposited under the number DSM ACC2249 at the German Collection ofMicroorganisms and Cell Cultures Ltd. (DSMZ) according to the BudapestTreaty and producing the antibody designated 4G8B4B12.

The invention further relates to a method for producing hybridoma cellssynthesizing and releasing an antibody directed against the nativeunmodified FLT3/FLK2 receptor protein. This method comprises the stepsgenerally known in the art, as described for example by Buhring et al.in Hybridoma 1991, Vol. 10, No. 1, pp. 77-78:

1. Immunizing or sensitization of an animal, preferably a mouse of theBalb/c line, with the antigen or immunogen;

2. collecting the antibody-producing cells, preferably the lymphocytesof the spleen of that animal;

3. fusion of these antibody-producing cells with a stable, immortalizedcell line, preferably a myeloma cell line, to hybridoma cells; and

4. isolation and multiplication (cloning) of such hybridoma cells thatsecrete an antibody binding to the antigen.

The method according to the invention is characterized by the fact thatthe animal is immunized with cells of the murine cell line BA/F3 thathad previously been transfected with the complete cDNA-sequence of thehuman FLT3/FLK2 receptor (BA/F3-huFLT3).

It is advantageous that the cells of this cell line BA/F3-huFLT3 presenta strong expression of FLT3/FLK2 receptor protein, as has been shownduring the experiments leading to these transfected BA/F3-huFLT3 cells.

When screening hybridoma cells producing antibodies specific forFLT3/FLK2 receptor protein, it is preferred, if only those isolated andcloned hybridoma cells are selected which produce antibodies having aspecificity for the transfected BA/F3-huFLT3 and showing a negativereaction with cells of the non-transfected BA/F3 cell line.

The invention also relates to the use of the inventive monoclonalantibody directed against the FLT3/FLK2 receptor protein for diagnosticand/or therapeutic treatment of tumors, particularly of malignanthematopoietic cells as for example lymphoid and myeloid leukemia cells.

Surprisingly, it has been found that most malignant hematopoietic cellsas, for example, lymphoid and myeloid leukemia cells comprise acomparably high content of FLT3/FLK2 receptor protein. An antibodyaccording to the invention coupled to a means for detection, forinstance a radioactive marker, indirectly binds this detection means tothe respective cells and thus allows the direct detection of the cells,for example using x-ray diagnostic/scintigraphic methods. Therefore, avery early diagnosis of tumors is possible, even in vivo.

In an analogous way the antibody may be coupled to a therapeuticallyactive agent and therefore allows a direct and targeted modulation oreven elimination of FLT3/FLK2 receptor protein carrying cells,particularly leukemia cells.

In order to facilitate the therapeutic and/or diagnostic application ofthe antibody according to the invention, the antibody may be mixed in apharmaceutical composition with adequate accessory substances.Consequently, the invention also relates to a pharmaceutical agent fordiagnostic and/or therapeutic treatment of tumors, comprising anantibody produced and released by the hybridoma cells deposited underNo. DSM ACC2249 at the German Collection of Microorganisms and CellCultures Ltd. (DSMZ).

Using an antibody according to the invention, cells carrying the humanFLT3/FLK2 receptor protein may be detected in a suspension of different(human) cells using the methods known in the art, for instance theenzyme-linked immunosorbent assay, ELISA, or the radio immuno assay,RIA. The present invention therefore also relates to a kit for thedetection of human FLT3/FLK2 receptor protein, comprising a monoclonalantibody produced by the hybridoma cells deposited under No. DSM ACC2249at the German Collection of Microorganisms and Cell Cultures Ltd.(DSMZ).

In connection with the present invention, it has surprisingly been foundthat the monoclonal antibody designated 4G8B4B12, produced by thehybridoma cells deposited under No. DSM ACC2249 at the German Collectionof Microorganisms and Cell Cultures Ltd. (DSMZ) binds to human stemcells.

The invention therefore also relates to the use of the antibody 4G8B4B12for detection of hematopoietic cells, as well as to a kit for thedetection of hematopoietic cells, comprising the antibody 4G8B4B12. Itis thereby possible to separate undifferentiated CD34⁺ subpopulationsand to select and purify cells from bone marrow for functional analyses.

Further advantages can be taken from the following description.

It is understood that the afore-mentioned features and those to beexplained below can be used not only in the specific combinations, butalso in other combinations or alone without going beyond the scope ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described hereafter with reference to certainapplications and embodiments, in combination with the drawings, inwhich:

FIG. 1 shows a histogram A of flow cytometer measurements of 4G1 cells,incubated with ligand and without antibody, and a histogram B of a flowcytometer measurement of 4G1 cells, incubated with ligand and antibody4G8B4B12 (therein designated 4G8); and

FIG. 2 shows three histograms of flow cytometer measurements on BV-173cells, preincubated at B with a control antibody and then incubated withthe antibody 4G8B4B12 (therein 4G8) and the ligand, at C directly withthe antibody 4G8B4B12 (therein 4G8) according to the invention and withthe ligand, and at A with the ligand alone.

DETAILED DESCRIPTION EXAMPLE 1

Generation and characterization of monoclonal antibodies directedagainst the FLT3/FLK2 receptor protein

Cells of a murine cell line BA/F3 that had previously been transfectedwith the complete cDNA for the human FLT3/FLK2 receptor protein(BA/F3-huFLT3) are used as antigen.

To generate these cells the complete coding sequence of the human cDNAfor FLT3/FLK2 receptor protein was cloned into the mammalian expressionvector pCD-SR and introduced together with a plasmid conferring G418resistance into the interleukin-3 dependent BA/F3 cells.

Transfected cells expressing the FLT3/FLK2 receptor were isolated andconcentrated on the basis of their G418 resistance and according totheir growth in the presence of recombinant FLT3/FLK2 receptorprotein-ligand (FL) (Lyman et al., Cell 75:1157, 1993). One clone, 4G1,was selected after limited dilution and tested for its ability toexpress FLT3/FLK2 receptor protein. For this purpose cells of clone 4G1were first labeled with radioactive methionine (³⁵ S) and then incubatedwith a rabbit antiserum directed against the inserted domain of thetyrosine kinase of the receptor protein FLT3/FLK2. The thus generatedimmunoprecipitate was analyzed using polyacrylamide gel electrophoresis.

The thus confirmed BA/F3-HuFLT3 cells of the clone 4G1 were used as theantigen.

Eight weeks old Balb/c mice are immunized intraperitoneally twice, atintervals of 10 days, with 10⁷ cells of the clone 4G1. Four days beforethe fusion, 5×10⁵ cells are administered directly into the spleen inorder to reinforce the immune response.

The formation of antibodies in the organism of the mouse is tested byscreening the blood serum of the respective animal for bindingproperties with the antigen using the ELISA test well-known to anyperson skilled in the art.

Approximately 3 weeks later the lymphocytes of the successfullyimmunized animal are collected by removing the animal's spleen anddisintegrating it into a cell suspension.

The suspended spleen cells are fused with myeloma cells of the knowncell line SP2/0 in the presence of polyethylene glycol. The fusionculture is cultivated in a medium containing hypoxanthine, aminopterineand thymidine (HAT), herein HAT-RPMI-1640, in which only hybrid cellsgrow as these have both the property of myeloma cells to divideinfinitely, and the property of the antibody-producing lymphocytes togrow in a medium containing HAT.

Following fusion, the cells are plated into microtiter plates and areincubated at 37 C in the presence of 5% CO₂.

The culture supernatants are screened in a flow cytometer after 10-14days on cells of the clone 4G1. In a second step the supernatants weretested for reactivity with the cells of the cell line BA/F3, as thesecells do not express antigens. Hybridoma cells producing antibodiesspecific for 4G1 cells are selected, isolated and cultivated, i.e.cloned, according to the known limited dilution method.

Positively reacting hybridoma cell cultures are subjected to furthercultivation, the antibodies are concentrated, purified andcharacterized.

The monoclonal antibody 4G8B4B12 was obtained using the above-mentionedscreening strategy. Using a phycoerythrine (PE)-conjugatedanti-isotype-specific antiserum by direct immunofluorescence using flowcytometry, the isotype IgG1 was determined.

Production, purification and characterization of the antibodies werecarried out using methods generally known in the art.

The antibody 4G8B4B12 produced by the hybridoma cells deposited underNo. DSM ACC2249 at the German Collection of Microorganisms and CellCultures Ltd. (DSM), exhibits the following characteristic features:

    ______________________________________                                        Immunoglobulin class: IgG1                                                                           specific binding affinity to: human FLT3/FLK2                                  receptor protein                                         (extracellular                                                                domain)                                                                    ______________________________________                                    

EXAMPLE 2

Identification of the antigen recognized by the monoclonal antibody4G8B4B12.

Identification of the antigen was carried out by testing binding tocells with and without FLT3/FLK2 receptor.

A sample (A) containing mouse fibroblasts of the cell line BA/F3-huFLT3generated according to example 1 and transfected with the human cDNA forFLT3/FLK2 receptor, was incubated with the antibody 4G8B4B12 of theinvention.

As a control, a sample (B) containing mouse fibroblasts ofnon-transfected cell line BA/F3 was equally incubated with the antibody4G8B4B12 in the same concentration as sample (A).

Both samples were labeled with an anti-IgG1-PE antiserum and thenanalyzed in a flow cytometer.

Binding of the antibody 4G8B4B12 could be shown exclusively for sample(A). This means that the antibody 4G8B4B12 according to the inventionhad specifically bound to those cells transformed with the cDNA forhuman FLT3/FLK2 receptor and therefore expressing the human FLT3/FLK2receptor.

EXAMPLE 3

Identification of the monoclonal antibody 4G8B4B12 as a non-antagonisticand slightly agonistic reacting antibody

In tyrosine kinase receptors such as the FLT3/FLK2 receptor receptoractivation initiated by ligand binding first leads to formation ofreceptor dimer complexes and their internalization into the cell. Someantibodies can simulate the ligand, i.e. they act agonistically, andtherefore lead to such a receptor dimer formation and internalization aswell (Buhring et al., Cancer Res. 53: 4424, 1993).

A sample (A) containing 4G1 cells (obtained according to example 1) wasincubated with 200 ng/ml biotinylated FLT3 ligand-biotine andstreptavidin-phycoerythrine (SA-PE).

A second sample containing 4G1 cells (B) was initially incubated with 7μg/ml of the antibody 4G8B4B12 for 30 minutes and subsequently treatedas sample (1A).

Both samples were analyzed in the flow cytometer. The resultinghistograms are given in FIG. 1.

Both histograms show equally strong signals. This means that theantibody 4G8B4B12 according to the invention has no inhibiting effect tothe ligand binding and therefore does not act antagonistically (Buhringet al., Cancer Res. 53: 4424, 1993).

For detection of the agonistic reaction a sample (B) containing cells ofthe hematopoietic pro-B cell line BV-173 (DSM ACC 20; freely accessible)was preincubated with a control IgG1 antibody for 2 hours at 37 C, thenincubated with the antibody 4G8B4B12 of the invention and finallyincubated with anti-IgG1-PE antiserum.

Another sample (C) was initially incubated with the antibody 4G8B4B12and subsequently treated as sample (B).

A control sample (A) was initially incubated with 100 ng/ml FLT3/FLK2receptor ligand and then treated as sample (B).

All three samples were analyzed in the flow cytometer. The resultinghistograms are given in FIG. 2. Compared to histogram B histogram Cshows a signal reduced by approximately 33%. This means that theantibody 4G8B4B12 according to the invention has caused internalizationof some FLT3/FLK2 receptors and therefore acts slightly in an agonisticway (ligand stimulating).

Compared to histogram B histogram A shows a signal reduced by 90% causedby internalization of the FLT3/FLK2 receptors by naturally occurringligands.

EXAMPLE 4

Use of the monoclonal antibody 4G8B4B12 for detection of subpopulationsof blood stem cells

A sample containing freshly obtained bone marrow cells was separated ina Ficoll-Hypaque density gradient and the normal, mononuclear bonemarrow cells were isolated and concentrated.

These mononuclear bone marrow cells were then incubated with aPE-conjugate of the antibody 4G8B4B12 of the invention and with knownfluorescein-isothiocyanate (FITC)-conjugated antibodies directed againstthe stem cell antigens HLA-DR, CD34 and CD62L, respectively, CD33occurring in myeloid cells, CD71 occurring in erythroid and progenitorcells, CD10 and CD19 occurring in B-cells, respectively and CD3 and CD7,occurring in T-cells, respectively.

The cells were analyzed in a flow cytometer.

The results of the analysis show:

a strong co-expression of FLT3/FLK2 with the stem cell markers CD34,HLA-DR and CD62L (55% and 100% and 60%, respectively),

a weaker co-expression of the myeloid cell marker CD33 and the erythroidand progenitor cell markers CD10 and CD19 (60% and 20% and 20%,respectively), and

no co-expression with the T-cell markers CD7 and CD3.

This means that blood stem cells as well as myeloid cells and progenitorcells of the B-line can be identified and particularly differentiatedfrom T-cells using the antibody 4G8B4B12 of the invention.

A 3-color analysis was carried out for characterization ofsubpopulations of the CD34⁺ stem cell population as follows:

The mononuclear cells were incubated with a PE-conjugate of the antibody4G8B4B12 of the invention, with a known FITC-conjugated antibodydirected against CD34, and a known biotine-conjugated antibody directedagainst CD117, and subsequently analyzed in the flow cytometer.

The obtained results show that 23% of the CD34⁺ cells also express thereceptor protein FLT3/FLK2 and 50% of the cells of this subpopulationadditionally comprise the protein CD117.

Analyses on CD34⁺⁺ and CD34⁺⁺⁺ cells show that the number of cells ofthe subpopulation of FLT3/FLK2⁺ /CD117⁺ cells decreases when CD34expression is decreasing (from CD34⁺⁺⁺ via CD34⁺⁺ up to CD34⁺), i.e.with ongoing development by the blood system cells, while thesubpopulation of FLT3/FLK2⁺ /CD117⁻ cells is increasing.

It is therefore possible to isolate two new subpopulations of the CD34-positive population of blood stem cells using the antibody 4G8B4B12of the invention, namely the subpopulation FLT3/FLK2⁺ /CD117⁺ and thesubpopulation FLT3/FLK2⁺ /CD117⁻.

EXAMPLE 5

Use of the monoclonal antibody 4G8B4B12 for detection of leukemic blastsof the myeloid and B-lymphocytic line

By centrifugation in a Ficoll gradient, a pure (>90%) population ofleukemic blasts was obtained from bone marrow or peripheral blood ofpatients suffering from leukemia.

The cells were classified according to the French-American-British (FAB)classification (Bennet et al., Ann. Intern. Med. 103: 620, 1985) andthis classification was confirmed by phenotypic analyses.

The cells were incubated with the antibody 4G8B4B12, the bound antibodywas labeled with the PE-conjugated anti-IgG1 antiserum and the cellswere analyzed in the flow cytometer.

In most of the blast-populations of patients suffering from acutemyeloid and/or B-lymphocytic leukemia a positive reaction and thereforethe presence of FLT3/FLK2 receptor protein was detected.

Further to these analyses using freshly isolated leukemia cells, samplesof megakaryoblastic cell lines, myeloid/monocytic cell lines, and B-celllines were tested.

The cells of all samples were equally incubated with the antibody4G8B4B12, the bound antibody was labeled with a PE-conjugated anti-IgG1antiserum and the cells were analyzed in the flow cytometer.

A positive reaction and therefore an expression of FLT3/FLK2 receptorprotein was found on most of the cell lines of the B-lymphocytic line,and on few of the myeloid line.

The antibody 4G8B4B12 of the invention has therefore proven to be aparticularly well-suited means for the detection and identification ofmalignant hematopoietic cells of the myeloid and B-lymphocytic line.

What is claimed is:
 1. A monoclonal antibody binding specifically to thehuman FLT3/FLK2 receptor protein and being produced and released byhybridoma cells that are deposited at the International DepositaryAuthority DSMZ-Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH Mascheroder Weg 1b, D-38124 Braunschweig, Germany, as of Dec. 19,1995 under No. DSM ACC2249 in accordance with the Budapest Treaty, anddesignated 4G8B4B
 12. 2. Hybridoma cells deposited at the InternationalDepositary Authority DSMZ-Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH Mascheroder Weg 1b, D-38124 Braunschweig, Germany, asof Dec. 19 1995 under No. DSM ACC2249 in accordance with the BudapestTreaty, and designated 4G8B4B
 12. 3. A composition comprising anantibody according to claim 1 and a pharmaceutically acceptable carrier.4. The composition according to claim 3, wherein the antibody is coupledto a labeling marker.
 5. Kit comprising an antibody according to claim1.